Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.

Summary Kinase Suppressors of Ras 1 and 2 (KSR1 and KSR2) function as molecular scaffolds to potently regulate the MAP kinases ERK1/2 and affect multiple cell fates. Here we show that KSR2 interacts with and modulates the activity of AMPK. KSR2 regulates AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryo fibroblasts and glycolysis in a neuronal cell line. Disruption of KSR2 in vivo impairs AMPK-regulated processes affecting fatty acid oxidation and thermogenesis to cause obesity. Despite their increased adiposity, ksr2-/- mice are hypophagic and hyperactive, but expend less energy than wild type mice. In addition, hyperinsulinemic-euglycemic clamp studies reveal that ksr2-/- mice are profoundly insulin resistant. The expression of genes mediating oxidative phosphorylation is also down regulated in the adipose tissue of ksr2-/- mice. These data demonstrate that ksr2-/- mice are highly efficient in conserving energy, revealing a novel role for KSR2 in AMPK-mediated regulation of energy metabolism.

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser 539 as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser 539 phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser 539 plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.The Hippo signaling pathway, originally defined in Drosophila, controls organ size, tumorigenesis, and cell contact inhibition by regulating cell proliferation and apoptosis (1-3). In mammalian cells, kinases Mst1/2 (orthologs of Drosophila Hippo) phosphorylate and activate Lats1/2 (orthologs of Warts) (4, 5). Lats, in turn, phosphorylates and inactivates the downstream effectors YAP/TAZ (orthologs of Yorkie) (6 -9). The transcriptional coactivators YAP and TAZ function together with transcription factors such as TEAD1-4 (Scalloped in Drosophila) (10 -13) to induce target gene expression, including Birc5 (8), cytokines such as connective tissue growth factor (10,14), and the EGF family member amphiregulin (15). Accumulated evidence suggests that this emerging signaling pathway plays a critical role in cancer development with the most evident contribution to hepatocellular carcinoma (1, 2, 16). For example, mice lacking Lats1 or WW45 (ortholog of Salvador) develop several types of tumors (17)(18)(19). Overexpression of YAP or loss of Mst1 and Mst2 in mouse liver dramatically increases the organ size and eventually induces hepatocellular carcinoma (8, 20 -23). The YAP locus is consistently amplified, and elevated YAP expression has been observed in many human cancers, including liver cancers (8, 14, 24 -26).KIBRA, a WW domain-containing protein (27), was originally identified as a memory performance-associated protein (28 -30). The physiological function of human KIBRA is not well understood, although it has been shown to play a role in podocyte migration (31, 32) and to be involved in age-dependent risk of Alzheimer disease (33). It was also reported to interact with discoidin domain receptor 1 to modulate collageninduced signaling (34). Interestingly, KIBRA expression wa...

Mitogen-activated protein kinase pathways are implicated in the regulation of cell differentiation, although their precise roles in many differentiation programs remain elusive. The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade has been proposed to both promote and inhibit adipogenesis. Here, we titrate expression of the molecular scaffold kinase suppressor of Ras 1 (KSR1) to regulate signaling through the Raf/MEK/ERK/p90 ribosomal S6 kinase (RSK) kinase cascade and show how it determines adipogenic potential. Deletion of KSR1 prevents adipogenesis in vitro, which can be rescued by introduction of low levels of KSR1. Appropriate levels of KSR1 coordinate ERK and RSK activation with C/EBP␤ synthesis leading to the phosphorylation and stabilization of C/EBP␤ at the precise moment it is required within the adipogenic program. Elevated levels of KSR1 expression, previously shown to enhance cell proliferation, promote high, sustained ERK activation that phosphorylates and inhibits peroxisome proliferator-activated receptor gamma, inhibiting adipogenesis. Titration of KSR1 expression reveals how a molecular scaffold can modulate the intensity and duration of signaling emanating from a single pathway to dictate cell fate.

In primary mouse embryo fibroblasts (MEFs), oncogenic Ras induces growth arrest via؊/؊ MEFs are completely resistant to Ras V12 -induced transformation. These data show that escape from senescence is not necessarily a precursor for oncogenic transformation. Furthermore, these data indicate that KSR1 is a member of a unique class of proteins whose deletion blocks both senescence and transformation.

Val-12 but had no effect on the ability of Ras Val-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development.

A chimeric growth factor receptor (CSF1R/IR) was constructed by splicing cDNA sequences encoding the extracellular ligand binding domain of the human colony stimulating factor-1 (CSF-1) receptor to sequences encoding the transmembrane and cytoplasmic domains of the human insulin receptor. The addition of CSF-1 to cells transfected with the CSF1R/IR chimera cDNA stimulated the tyrosine phosphorylation of a protein that was immunoprecipitated by an antibody directed against the carboxyl terminus of the insulin receptor. Phosphopeptide maps of the 32 P-labeled CSF1R/IR protein revealed the same pattern of phosphorylation observed in 32 P-labeled insulin receptor ␤ subunits. CSF-1 stimulated the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and Shc in cells expressing the CSF1R/IR chimera. Lipid accumulation and the expression of a differentiation-specific marker demonstrated that 3T3-L1 preadipocytes undergo CSF-1-dependent differentiation when transfected with the CSF1R/IR chimera cDNA but not when transfected with the expression vector alone. A 12-amino acid deletion within the juxtamembrane region of the CSF1R/IR (CSF1R/IR⌬960) blocked CSF-1-stimulated phosphorylation of IRS-1 and Shc but did not inhibit CSF-1-mediated differentiation of 3T3-L1 preadipocytes. These observations indicate that adipocyte differentiation can be initiated by intracellular pathways that do not require tyrosine phosphorylation of IRS-1 or Shc.A primary goal in the study of insulin action is the identification of the intracellular pathways that lead ultimately to changes in the rates of growth, development, and metabolism in primary target tissues of insulin (e.g. adipose, muscle, and liver). One strategy for the study of insulin-sensitive intracellular signaling has been to identify structural features within the insulin receptor that are important components of divergent signal transduction pathways. The goal of this strategy has been to create mutations within specific receptor sequences that result in the selective disruption of some insulin-regulated metabolic pathways while leaving others intact. Deletion mutagenesis of the insulin receptor cytoplasmic domain has generated insulin receptors with altered biological properties (1-4). Experiments with these receptor mutants have led to the suggestion that different regions of the insulin receptor cytoplasmic domain may play roles in modulating the distinct biological effects of insulin. Most mutations of the insulin receptor cytoplasmic domain have removed or altered autophosphorylation sites within the cytoplasmic domain (5-8). This approach has furthered the notion that tyrosine phosphorylation and the tyrosine kinase encoded within the receptor ␤ subunit are essential components of normal insulin action. The majority of structure/function analyses of the insulin receptor, however, have been performed in cells expressing low levels of endogenous insulin receptors, e.g. Chinese hamster ovary (CHO) 1 or Rat-1 fibroblasts cell lines (reviewed in Ref. 9). Although su...

KSR (kinase suppressor of Ras) has been proposed as a molecular scaffold regulating the Raf/MEK/ERK kinase cascade. KSR is phosphorylated on multiple phosphorylation sites by associated kinases. To identify potential mechanisms used by KSR to regulate ERK activation, green fluorescent protein was fused to intact and mutated KSR constructs lacking specific phosphorylation sites, and the subcellular distribution of each construct was observed in live cells. Mutation of a subset of KSR phosphorylation sites caused the redistribution of KSR to the nucleus. To determine whether intact KSR is normally imported to the nucleus, REF-52 fibroblasts expressing KSR were treated with 10 nM leptomycin B, which inhibits Crm1-dependent nuclear export. KSR accumulated in the nucleus within 2 h of treatment with leptomycin B, suggesting that KSR cycles continuously through the nucleus. Nuclear import of KSR was blocked by mutations that inhibit the interaction of KSR with MEK. Coexpression of fluorescent forms of KSR and MEK in cells revealed that each protein promoted the localization of the other in the cytoplasm. These data indicate that the subcellular distribution of KSR is dynamically regulated through phosphorylation and MEK interaction in a manner that may affect signaling through ERK.